Maintaining effective immune surveillance without provoking autoimmune reactions requires the precise titration of effector T cell responses. Autoimmune disorders arise when the immune system mounts an immune response against self-antigens (see, e.g., Ludewig et al. (1999) Immunol Rev. 169:45-54). While the mechanisms involved in the triggering and maintenance of autoimmune reactions is unclear, it is likely that the appearance of previously immunologically ignored antigens in secondary lymphoid organs is involved.
Dendritic cells are bone-marrow derived antigen presenting cells (APCs) that play a key role in the immune response (see, e.g., O'Neill et al. (2004) Blood 104:2235-2246). DCs internalize bacteria, viruses, dying cells, and various complex molecules through phagocytosis, endocytosis, and pinocytosis. Incorporated proteins are broken down into peptides, which are then presented on the DC cell surface along with MHC class I and class II molecules. Antigens loaded onto MHC class I are typically derived from endogenous proteins and are recognized by CD8+ T cells, whereas MHC class II loaded antigens are generally derived from external proteins and are recognized by CD4+ T cells. Following antigen capture, immature DC cells mature to form mature DCs which show reduced phagocytosis, migrate to lymphoid tissues, and have enhanced T cell stimulation capacity.
In lymphoid tissues, DCs prime naïve T cells, stimulating their clonal expansion and differentiation, and can also interact with B cells and cells of the innate immune system, including NK cells. Activated NK cells can kill immature, but not mature, DC cells. As antigen transport and primary sensitization of T lymphocytes is mainly mediated by antigen presenting dendritic cells, it is likely that the inappropriate presentation of self antigens by dendritic cells contributes at least in part to autoimmune disorders.
Natural killer (NK) cells are a subpopulation of lymphocytes involved in non-conventional immunity. NK cells provide an efficient immunosurveillance mechanism by which undesired cells such as tumor or virally-infected cells can be eliminated. NK cell activity is regulated by a complex mechanism that involves both activating and inhibitory signals (see, e.g., Moretta et al. (2001) Annu Rev Immunol 19:197-223; Moretta et al. (2003) EMBO J EPub December 18; Ravetch et al. (2000) Science 290:84-89; Zambello et al. (2003) Blood 102:1797-805; Moretta et al. (1997) Curr Opin Immunol 9:694-701; the entire disclosures of which are herein incorporated by reference).
Several distinct NK-specific receptors have been identified that play important roles in the NK cell mediated recognition and killing of HLA Class I deficient target cells. These receptors, termed NKp30, NKp46 and NKp44, are members of the Ig superfamily. Their cross-linking, induced by specific mAbs, leads to a strong NK cell activation resulting in increased intracellular Ca++ levels, triggering of cytotoxicity, and lymphokine release. Importantly, mAb-mediated activation of NKp30, NKp46, and/or NKp44 results in an activation of NK cytotoxicity against many types of target cells. These findings provide evidence for a central role of these receptors in natural cytotoxicity.
NK cells are negatively regulated by major histocompatibility complex (MHC) class I-specific inhibitory receptors (Kärre et al. (1986) Nature 319:675-8; Ohlen et al, (1989) Science 246:666-8). These specific receptors bind to polymorphic determinants of major histocompatibility complex (MHC) class I molecules or HLA and inhibit natural killer (NK) cell lysis. In humans, certain members of a family of receptors termed killer Ig-like receptors (KIRs) recognize groups of HLA class I alleles (see, e.g., Yawata et al. (2002) Crit Rev Immunol 22:463-82; Martin et al. (2000) Immunogenetics. 51:268-80; Lanier (1998) Annu Rev Immunol. 16:359-93; the entire disclosures of which are herein incorporated by reference).
Another important inhibitory receptor on NK cells is CD94-NKG2A, which interacts with the non-classical MHC class 1 molecule HLA-E (see, e.g., Braud et al. (1998) Nature 391:795-799; Lee et al. (1998) PNAS 95:5199-5204; Vance et al. (2002) PNAS 99:868-873; Brooks et al. (1999) J Immunol 162:305-313; Miller et al. J Immunol (2003) 171:1369-75; Brooks et al. (1997) J Exp Med 185:795-800; Van Beneden et al. (2001) 4302-4311; U.S. patent application no. 20030095965; the entire disclosures of each of which are herein incorporated by reference). Some of these receptors have the capacity to modulate thresholds of T cell antigen receptor-dependent T cell activation. In the rare absence of inhibitory receptors, the activating isoforms may augment T cell effector functions and contribute to autoimmune pathology. The amino acid sequence of NKG2A varies among mammals, including among primates. For example, the human and rhesus monkey versions of the NKG2A proteins share less than 90% identity, including approximately 86% within the ligand binding domain.
Efforts towards therapeutics for modulating NKG2A, essentially for the prevention of inflammation, have focused on the study of the nonclassical MHC class I molecules, HLA-E for the human receptor and Qa-1b for the mouse receptor. For cell surface expression, these MHC molecules preferentially bind peptides derived from the signal peptides of other MHC class I molecules. The expression of other class I MHC molecules can regulate the expression of HLA-E, thereby allowing NK cells to monitor the state of the MHC class I dependent antigen presentation pathway in potential target cells. The level of cell surface HLA-E is critical for the NK cell cytotoxicity towards tumor and virally infected cells. Therapeutic strategies for modulating HLA-E expression or function have generally been directed towards using HLA-I or HSP60 peptides to induce a protective state for the prevention of inflammation such that NK cells are not activated.
United States patent publication 20030095965 discloses an antibody, 3S9, that binds to NKG2A, NKG2C and NKG2E. 3 S9 purportedly causes cross-linking of those receptors and concomitant inhibition of NK cell-mediated lysis. Co-owned PCT patent publication WO 2005/105849 discloses the use of an antibody that specifically binds to an NK receptor, including NKG2A, to treat a patient suffering from NK-type lymphoproliferative disease of granular lymphocytes (NK-LDGL). Such antibodies inhibit NK cell activity.
Monoclonal antibodies have proven to be enormously useful for the diagnosis and treatment of various diseases. Therapeutic monoclonal antibodies can act through different mechanisms. Some antibodies, such as Rituxan, recognize antigens (CD20 in the case of Rituxan) present on the surface of pathological cells, e.g., tumor cells, and act by directing the immune system to destroy the recognized cells. Other antibodies, such as Bexxar, Oncolym, or Zevalin, are coupled to radioisotopes, chemotherapeutic agents, or toxins, leading to the direct killing of cells bound by the antibodies. Still others, such as Basiliximab and Daclizumab (which block IL-2), the IgE blocking Omalizumab, and efaluzimab, act to block the activity of specific proteins. Antibody based therapies are well known in the art and are reviewed, e.g., in Gatto (2004) Curr Med Chem Anti-Canc Agents 4(5):411-4, Casadevall et al. (2004) Nat Rev Microbiol. 2(9):695-703, Hinoda et al. (2004) Cancer Sci. 95(8):621-5, Olszewski et al. (2004) Sci STKE. July 06(241):pe30, Coiffier (2004) Hematol J. Suppl 3:S154-8, Roque et al. (2004) Biotechnol Prog. 20(3):639-54, the entire disclosures of each of which is herein incorporated by reference.
Before antibodies can be used for therapeutic applications in humans, or enter clinical trials, they must go through pre-clinical studies in non-human animals to assess various parameters such as their toxicity, in vivo efficacy, bioavailability, half-life and various other pharmacokinetic and pharmacodynamic parameters. Such assays are typically carried out in mammals, and, preferably, where they have biological activity, i.e. where the mAb is reacting to the homolog molecule in the specie, therefore where one can expect the greatest physiological similarity to humans. However, studies in nonhuman primates can be impeded if an antibody directed against a human protein does not bind to the nonhuman animal homolog of the target protein. When crossreactivity is present, in contrast, not only can the in vivo efficacy of the antibody be tested in the animal, but other issues such as side effects, toxicity, or kinetic properties that are related to the binding of the antibody to the target protein can be studied as well. Examples of readily available primates include the New World monkeys and Old World monkeys, such as the cynomolgus monkey (Macaca mulatta), the rhesus macaque (Macacus mulatta), the African green monkey (Chlorocebus aethiops), the marmoset (Callithrix jacchus), the saïmiri (Saimiri sciureus), all available from “Centre de Primatologie” (CDP:ULP, Fort Foch, 67207 Niederhausbergen, France), and the baboon (Papio hamadryas) available from “Station de Primatologie du CNRS”, CD56, 13790 Rousset/Arc, France). Chimpanzees and apes in general may also be used for testing a candidate medicament, although such instances are rare and generally only when no other alternative for testing exists or has been exhausted.
As antibodies bind to specific 3-dimensional features of their targets, slight changes in the amino acid sequence of a target protein can abolish binding altogether, making it unpredictable whether a given antibody directed against a protein from one species will also bind to homologous proteins sharing some but not complete sequence identity. Many instances have been described in which antibodies directed against a human protein, for example, do not bind to homologs in even closely related species. For example, some antibodies against the human CD4 protein do not bind to monkey homologs, even though the human and rhesus monkey CD4 proteins share close to 94% percent identity (see, e.g., Genbank IDs GI:116013 and 20981680; Sharma et al. (2000) JPET 293:33-41, 2000, the entire disclosures of which are incorporated herein by reference). Other examples include some antibodies against human CD3, a widely pursued pharmaceutical target for antibody development; antibodies, for example UCHT2, otherwise having properties suitable for development do not crossreact with the monkey CD3 protein.
In view of the prominence and severity of many autoimmune disorders, and the role of mature dendritic cells in coordinating the immune response against self-antigens, there is a great need in the art for new and effective therapies that modulate the activity or level of dendritic cells underlying such disorders. Moreover, there is a need for therapies against disorders characterized by aberrant cells (e.g., certain cancer or virally infected cells) that are able to shield themselves from destruction by the immune system. Finally, there is also a need to find a valid in vivo test system for the therapeutic potential in humans of monoclonal antibodies against NKG2A. The present invention addresses this and other needs.